This invention relates to cDNAs which encode Ndr2-related proteins and to the use of the cDNAs and the encoded proteins in the diagnosis and treatment of cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Reducing agents and tunicamycin-responsive protein (RTP; also referred to as Drgl, Cap43, and rit43) may play roles in atherosclerosis, tumorigenesis, differentiation, hypoxia, and cellular responses to stress (Agarwala et al. (2000) Biochem Biophys Res Commun 272:641-647). Human RTP is a 43 kDa cytoplasmic protein of 394 amino acids in length and is expressed ubiquitously. Although RTP is present predominantly in the cytoplasm of cells, immunofluorescence studies show that a fraction of RTP is located in the nucleus, an indication that RTP may be a signaling protein that shuttles between the cytoplasm and nucleus. The expression level of RTP increases in response to various chemical compounds, including homocysteine, cysteine, mercaptoethanol, tunicamycin, lysophosphatidylcholine, nickel compounds, okadaic acid, calcium ionophore, DNA-damaging agents, 1,25-(OH)2 vitamin D3, synthetic retinoids, and phorbol myristate acetate. RTP expression is decreased in tumor cell lines and in colon, breast and prostate tumors (Kurdistani et al. (1998) Cancer Res 58:4439-4444; van Belzen et al. (1997) Lab Invest 77:85-92). Overexpression of RTP inhibits the growth of cancerous cells. RTP expression is upregulated in human myelomonocytic cells by retinoids that inhibit proliferation and promote differentiation (Piquemal et al. (1999) Biochim Biophys Acta 1450:364-373) and in prostate adenocarcionma cells by androgens that induce differentiation (Ulrix et al. (1999) FEBS Lett 455:23-26). The activity of RTP may be regulated by phosphorylation. RTP is phosphorylated in vivo by an unknown kinase at multiple sites and can be phosphorylated by protein kinase A in vitro (Agarwala et al., supra). RTP is dephosphorylated in cells exposed to homocysteine. RTP expression levels change at different stages of the cell cycle. RTP expression is highest at the G1 and G2-M stages and lower at S phase (Kurdistani et al., supra). The transcription factor p53, a known tumor suppressor, induces expression of RTP. P53 causes the arrest of cell growth at G1 and G2, and RTP may play a role in this growth-arrest pathway.
Mouse Ndr1 is a homolog of RTP that was identified in a screen for genes regulated by N-myc. Ndr1 shows increased expression in N-myc deficient mice (Okuda and Kondoh (1999) Mech Dev 83:39-52). Myc family transcription factors control genes expressed during embryogenesis, proliferation, differentiation, apoptosis, and tumorigenesis (Grandori et al. (2000) Annu Rev Cell Dev Biol 16:653-699). N-myc expression is high during cell proliferation and decreased during cell differentiation. Rearrangements and mutations of Myc proteins are found in many tumors. N-Myc represses transcription of Ndr1. During mouse embryogenesis, Ndr1 expression is correlated with a decrease in N-myc expression and Ndr1 levels increase when cells begin to differentiate. Ndr1 appears, therefore, to be a signaling molecule involved in cellular differentiation.
Two Ndr1-related genes, Ndr2 and Ndr3, have recently been discovered (Okuda and Kondoh (1999) Biochem and Biophys Res Commun 266:208-215). The amino acid sequences of Ndr2 and Ndr3 proteins show 54% and 64% identity to Ndr1, respectively, and therefore are grouped with Ndr1 as the Ndr family. All three Ndr genes are expressed during embryogenesis, but at different times and show differences in the regulation of their expression levels. Unlike Ndr1, Ndr2 expression is not upregulated in N-myc deficient mouse embryos. Ndr1 expression, in mouse embryos, increases after 13.5 dpc whereas Ndr2 expression increases earlier, at 11.5 dpc, and Ndr3 expression is high at 9.5 dpc and shows little increase at later developmental stages. RTP, Ndr1, Ndr2, and Ndr3 may represent a gene family with differentiation-related functions.
The discovery of cDNAs encoding Ndr2-related proteins satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney.
The invention is based on the discovery of cDNAs encoding Ndr2-related proteins (NRP) which are useful in the diagnosis and treatment of cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney.
The invention provides an isolated cDNA comprising a nucleic acid sequence encoding a protein selected from the group consisting of the amino acid sequences of SEQ ID NO:1 (NRP1) and SEQ ID NO:2 (NRP2). The invention also provides an isolated cDNA or the complement thereof selected from the group consisting of the nucleic acid sequences of SEQ ID NO:3 and SEQ ID NO:11, a fragment of SEQ ID NO:3 selected from SEQ ID NOs:4-10 or a fragment of SEQ ID NO:11 selected from SEQ ID NOs:12-15, and a variant of SEQ ID NO:3 or SEQ ID NO:11 selected from SEQ ID NOs:16-31. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding NRP. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make NRP. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding NRP. The invention additionally provides a fragment, a variant, or the complement of the cDNA selected from the group consisting of SEQ ID NOs:2-31. In one aspect, the invention provides a substrate containing at least one of these fragments or variants or the complements thereof. In a second aspect, the invention provides a probe comprising a cDNA or the complement thereof which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single-stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney. In another aspect, the cDNA or a fragment or a variant or the complements thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a variant or the complements thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, a variant having at least 97% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, an antigenic epitope of SEQ ID NO:1 or SEQ ID NO:2, and a biologically active portion of SEQ ID NO:1 or SEQ ID NO:2. The invention also provides a composition comprising the purified protein in conjunction with a pharmaceutical carrier. The invention further provides a method of using the NRP to treat a subject with cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney.
The invention provides a method of using a protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney.
The invention also provides a method of using a protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which binds specifically to a protein which is expressed in cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney. The invention also provides a method of using an antibody to diagnose cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney. The invention further provides a method of using an antibody to treat cancer, particularly cancers of the intestine, breast, uterus, liver, brain, and kidney comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.
The invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:3-31, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.